Important notice regarding the use of Qiagen Biorobot (EZ1/Geno-M6/M48/M96).Various MLPA users have reported problems when using DNA extracted by the Qiagen EZ1 or M48 robot. If you are using a Qiagen Biorobot with your blood samples, use this supplementary protocol from Qiagen.

NEW: The 5 µl DNA sample used for MLPA reactions should contain a minimum of 5 mM Tris-HCl with a pH between 8 and 8.5. DNA samples diluted with water may give aberrant results due to depurination in the 5 minute 98 °C DNA denaturation step. In case the ionic composition of the sample is not known, we recommend to add Tris-HCl pH 8.5 to the sample, resulting in a 5-10 mM concentration.

In this Sample treatment section, you can find information about the following:

Although the MLPA reaction is robust and reproducible, the origin and treatment of sample DNA (tissue, extraction method, buffer used for elution and dilution, storage) can influence the MLPA peak pattern. Contaminants which remain after extracting or dissolving the DNA can affect DNA denaturation and/or amplification of certain MLPA probes. Contamination can be derived from the tissue used (heparin, melanin, haem compounds) or can be due to the extraction method used such as the high salt concentrations present in samples purified by the Qiagen EZ1 BioRobot.

Within a single experiment, one should preferably only compare samples that were extracted from the same tissue, by the same method and by the same laboratory. As the effect of contaminants is generally consistent and reproducible, a bias caused by impurities will often be corrected for by using similar amounts of each sample DNA.

When contaminants are present in large quantities or are present in different concentrations in different samples, problems may arise. If you are working with older samples, samples that have been extracted using different methods or with samples which may contain contaminants, try adding 10 mM Tris-HCl pH 8.5 to each sample and use only 50 ng DNA, or subject each sample to an extra purification step such as a simple ethanol precipitation.

1. Sample quality

To minimise the effects of contaminants, always use sample and reference DNA extracted with the same method and derived from the same source. For instance, when testing DNA extracted from FFPE breast tumour tissue, compare this to similarly extracted FFPE samples from healthy breast tissue (the sample does not have to be from the same individual).

We recommend using a total quantity of 50-250 ng of (human genomic) sample DNA in a 5 µl volume for each MLPA reaction. In case of problems that might be due to sample quality, use only 20-50 ng sample DNA. We recommend to use similar DNA concentrations to minimize variation caused by possible contaminants.

The 5 µl sample DNA used in MLPA should contain 10 mM Tris buffer with a pH between 8 and 8.5. DNA samples eluted, dissolved or diluted in water have insufficient buffering capacity. As a result, the sample DNA is rapidly degraded by depurination when heated at 98 °C in the first step of the MLPA procedure. This can affect the MLPA results. We recommend dissolving or diluting the sample DNA with TE. Also for long term storage, DNA is more stable in TE than in water.

In case it is not known whether the DNA is dissolved in water or in TE, addition of 5 or 10 mM Tris-HCl with a pH between 8.0 and 8.5 is recommended. You can do this by adding 1 µl 50 mM Tris-HCl pH 8.5 to a 4 µl sample DNA. We expect that this will diminish variation in results between samples purified by different methods or between samples received from different laboratories.

Long term (years) storage at 4 °C and repeated freeze-thaw cycles may influence sample quality and MLPA results. Evaporation can result in high EDTA concentrations and metabolites produced by molds may be potential PCR inhibitors.

MLPA users reported that reference samples and positive samples that were used for a prolonged time started showing aberrant results. We therefore recommend to aliquot regularly-used reference DNA samples and precious positive DNA samples and to store these at -20 °C. After thawing, the vial in use can be stored at 4 °C for at least 6 months.

Since MLPA probes recognize targets of only 50-80 nt long, MLPA is hardly influenced by DNA fragmentation. DNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples can thus be used. Please see the advice below on how to extract DNA from these tissues. As the FFPE derived DNA is not only fragmented but is often also damaged by depurination, cross-links and base modifications, the results obtained on FFPE derived DNA usually show a higher standard deviation as with normal sample DNA.

Contaminants:

Contaminants that do not influence MLPA

Some contaminants in DNA samples, such as proteins, do not influence MLPA. Therefore, MLPA can be used directly on lysates obtained by ProteinaseK digestion of cells or on DNA from FFPE samples.

Contaminants causing incomplete sample DNA denaturation

The concentration of salts in DNA samples should be low in order to allow DNA denaturation of those genomic regions that have a very high CG content. CpG islands with a GC content above 80% will not be completely denatured in samples that contain 40 mM NaCl or KCl, or more than 80 mM Tris-HCl.

The presence of other ions, such as Fe and Mg, can already cause denaturation problems at concentrations below 1 mM. A concentration of as little as 0.1 mM MgCl2 in the sample can even increase the denaturation temperature (Tm) by 12 °C (Eichhorn, 1962, Nature 194:474-5). For this reason, sample DNA that is dissolved or diluted in 1x PCR buffer cannot be used.

Incomplete DNA denaturation can be easily detected by low signals of the 88 and 96 nt D-control fragments. It results in very low signals of probes located close to CpG islands. Even signals of probes detecting a sequence located at a distance of 5-10kb from a strong CpG island can be reduced.

Contaminants inhibiting the PCR:
The MLPA PCR reaction is more sensitive to certain impurities than conventional PCR. The following can influence the PCR reaction:

PCR inhibitors derived from certain tissues that co-purify with DNA include haem, heparin and melanin. Heparin can be present in DNA purified from blood that is treated with heparin as anticoagulant, but is also naturally present in many tissues such as lung, intestine, renal, liver and other tissues containing mast cells. Melanin is present in pigmented cells such as skin and hair cells.

PCR inhibitors that can be present in a DNA sample as a result of the DNA purification method used include phenol/trizol remnants, SDS and Fe containing magnetic beads. Laboratories which experienced problems with DNA samples reported later that these could be attributed to left-over phenol and ethanol remnants.

DNA samples should not contain more than 1.5 mM EDTA as EDTA binds magnesium. A sufficiently high concentration of unbound magnesium (present in Ligase buffer B) is required for the ligation and PCR reaction. High EDTA concentrations that affect the MLPA PCR reaction can be present in DNA samples that have been concentrated by evaporation or SpeedVac.

The mode of action for many PCR inhibitors is reduction of polymerase activity. Not all MLPA probes react the same way to this reduced polymerase activity. While most probes will not be affected, some may show a reduction in peak height whereas yet others will increase in relative signal.

RNA contamination of samples
A minority of MLPA probes is sensitive to RNA contamination. These are probes that are present in very abundant RNAs. Examples include probes for the HBA and HBB genes (Blood derived DNA samples) and probes for (mitochondrial) ribosomal RNA genes. Depending on the probe orientation, the RNA molecules compete with probes for binding to the sample DNA or the probe oligonucleotides are sequestered by binding to the RNA. The P102 HBB, P140 HBA and P125 Mitochondrial DNA probemixes contain a protocol for RNAse digestion of DNA samples. For other products we currently have no evidence that RNAse treatment improves results.

In case of sample DNA contamination

Use less sample DNA (20-50 ng): when the DNA concentrations are quite high you may choose to strongly dilute your samples, thereby also diluting the concentration of contaminants. Make sure you use at least 20 ng DNA. The Q fragments present in each probemix at 64-70-76-82 nt indicate the amount of sample DNA present. When the Q fragment peaks are higher than 1/3 of the height of the 92 nt control peak, the amount of sample DNA is insufficient for reliable MLPA results.

Add 10 mM Tris-HCl pH 8.5 to each sample.

Perform an extra purification step: clean contaminated samples by ethanol precipitation or silica columns. This is especially a good idea for old samples if enough DNA is available. More information on purification can be found here.

When the MLPA reaction is properly performed and analysed, the probe standard deviation between samples should be a maximum of 10 % for the great majority of probes. When a higher variation is found, this may indicate that differences between samples exist. For variation due to electrophoresis problems please read the General MLPA protocol and the various files on the MLPA procedure that are present on our website.

2. Dissolving and diluting DNA samples

The buffer used to dissolve and/or dilute DNA is important. The pH of the DNA preparation should be between 8.0 and 8.5 in order to prevent depurination during the initial heat treatment at 98 °C.

We recommend the use of TE (10 mM Tris-HCl pH 8.2 + 0.1 mM EDTA) for dissolving and diluting DNA. Do not use more than 1.5 mM EDTA in the sample solution as it will bind Magnesium and may influence the ligation or PCR reaction!
DNA with insufficient buffering capacity, such as DNA samples diluted in water, are rapidly degraded by depurination when heated at 98 °C. This can affect the MLPA results.

The use of 1 x PCR buffer to dissolve or dilute DNA is also strongly discouraged! This buffer contains both Magnesium and salt, the combination of which prevents the denaturation of CG-rich areas. Our test results have shown that when the DNA is dissolved in 1 x PCR buffer (containing 50 M KCl, 1.5 mM MgCl2), most CpG islands are no longer completely denatured by a heat treatment of 98 °C. As a result, probes that recognize a sequence located less than 5-10 Kb from CpG islands may show a reduced probe signal (false positive deletion).

3. DNA extraction

MLPA does not require a special for DNA extraction method or kit. Many users obtain good results with simple salting out methods or by phenol/chloroform extraction. Various Qiagen kits are also widely used with good results. Please note that when DNA is eluted in water, Tris-HCl with a pH between 8.0 and 8.5 should be added to the sample (10 mM final concentration) before the 5 minutes 98 °C DNA denaturation treatment.

Problems with automated DNA extraction methods, such as Roche Magnapure and Qiagen’s EZ1 robot have been reported. It seems that the first method has been improved, as we now receive fewer comments about it. The standard protocol of the Qiagen EZ1 BioRobot and M48 Biorobot leaves too much salt in the samples, causing MLPA probes that detect sequences located in or close to a CpG island to become variable. Please read our recommendations at the start of this article. DNA extracted from heparin-treated blood can also give problems as heparin is difficult to remove from DNA preparations. We recommend the use of EDTA treated blood. You can find information about some suitable extraction methods in the MLPA general protocol.

Please note that at MRC-Holland, we only have little exprience with the various DNA extraction methods. MRC-Holland also has not tested MLPA on DNA derived from dried blood spot (DBS) cards, and the use of dried blood spot cards in combination with MLPA has not been approved for in vitro diagnostic use. Please e-mail us at info@mlpa.com about your experiences so that we can continue to improve our guidelines.